For many species of microorganisms, invasion and survival within mammalian cells is central to establishing a successful host-parasite relationship. The invasive character of pathogens, while deleterious to the health and viability of host cells, does provide a mechanism for transfer of molecules and aggregates across an intact cellular membrane. Thus, microorganisms having an attenuated invasive phenotype which minimizes the deleterious effect of infection through invasion are useful as a vaccine for the pathogen itself or as a vehicle for transporting molecules of interest into a host organism or its cells.
For example, a microorganism having attenuated invasiveness can provide for the transfer of genetic material into a mammalian host or its cells. In this manner, novel genetic capabilities can be imparted to the host. One capability of interest is the synthesis of surface membrane proteins or envelope proteins of pathogens. These proteins can then serve as antigens to provide a strong immune response, without the host having to suffer the effects of infection by the pathogen.
A number of microorganisms are known to invade mammalian cells including Yersinia, Shigella and Salmonella. The invasion of epithelial cells by Yersinia pseudotuberculosis is reported by Bovallius and Nilson, (1975) Can. J. Microbiol. 21:1997-2007 and Bolin et al., (1982) Infect. Immun. 37:506-512. Isberg and Falkow, (1985) Nature 317:262-264 and Isberg et al., (1987) Cell 50:769-778 describe the invasion locus of Yersinia pseudotuberculosis. Falkow et al., Reviews of Infectious Diseases, 9 Supp. 5 S450-S455 (1987) describes the Yersinia gene inv. Miller and Falkow, (1988) Inf. and Imm. 56:1242-1248 describe a second invasion gene named ail (for attachment invasion locus). Miller et al., (1989) Science 243:916-922 describe factors involved with virulence of bacterial pathogens. The nucleotide sequence of the Y. enterocolitica ail gene is described in Miller et al., (1990) Bacteriology 172:1062-1069. See also Miller et al., (1989) Infect. and Immun. 57:121-131.
The factors associated with Shigellae invasiveness are described by Hale et al., (1983) Infect. Immun. 40:340-350. Sansonetti et al., (1983) ibid 39:1392-1402 and Maurelli et al., (1985) ibid 49:164-171 describe the manipulation of the plasmid in Shigellae encoding functions essential for invasiveness.
Salmonella penetrate the intestinal epithelial barrier during infection. Takeuchi, A. (1967) M. J. Pathol. 50:109-136; Worton, K. J., et al. (1989) J. Med. Microbiol. 29:283-294. The interactions between Salmonella and the non-phagocytic cells lining the mucosal surface of the bowel, however, can be studied experimentally since Salmonella enter cultured epithelial cells in vitro. Finlay, et al. (1988) J. Cell Biol. 107:221-230; Finlay, et al. (1988) J. Infect. Dis. 162:1096-1106. In one laboratory strain of Salmonella typhimurium, which had undergone many genetic manipulations and was found to be non-invasive, complimentation of the defect by genes from a virulent strain led to the identification of a locust comprising of seven genes that is required for bacterial invasion and virulence. Galan, J. et al. (1989) Proc. Natl. Acad. Sci USA 86:6383-6387. Finlay et al., Science 243:940-943 describe an invasion gene of Salmonella.
Many attempts have been made to clone Salmonella genes into E. coli and to confer the ability to enter. epithelial cells. Such an approach was successful in identifying invasion factors in Yersinia species. Isberg and Falkow (1985), ibid. and Miller and Falkow (1988), ibid. Thus far, only one locus, from Salmonella typhi, has been identified by this approach. Elsinghorst, E. A., et al. (1989) Proc. Natl. Acad. Sci. USA 86:5173-5177. In addition to the aforementioned genetic factors, it has been determined that Salmonella invasiveness is regulated by oxygen such that bacteria grown aerobically are less invasive than bacteria grown under oxygen-limiting conditions. Lee and Falkow (1990) Proc. Natl. Acad. Sci. USA 87:4304-4308; Ernst et al. (1990) Infect. Immun. 58:2014-2016; and Schiemann, D. A. et al. (1991) Infect. Immun. 59:437-440.
In addition, St. Geme, J. W. et al. (1990) Infect. and Immun. 58, 4036-4044 describe the adherence and invasion of Haemophlilus influenzae into human epithelial cells. For a review of bacterial invasive strategies, see Finlay and Falkow (1989) Microbiological Reviews 53:210-230.